FUNCTION OF AN INTERFERON INDUCED UBIQUITIN HOMOLOG

干扰素诱导的泛素同系物的功能

• 批准号: 2184840
• 负责人: ARTHUR L HAAS
• 金额: $ 19.91万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184840
• 关键词: antisense nucleic acid; chemical binding; chemical conjugate; chemical stability; circular dichroism; covalent bond; enzyme mechanism; fluorescence; gene induction /repression; interferons; intermediate filaments; molecular cloning; posttranslational modifications; protein purification; protein sequence; protein structure function; recombinant proteins; tissue /cell culture; ubiquitin

DESCRIPTION (adapted from the applicant's abstract): Cell line specific effects of the interferons are mediated through the coordinated induction of a subset of genes expressed at precise times during the cytokine response. The principal investigator has shown that p15, a 17 kDa early gene product of type 1 interferon induction, bears marked homology to a tandem di-ubiquitin sequence. Subsequent evidence suggests the biological response of this Ubiquitin Cross Reactive Protein (UCRP) is mediated through covalent ligation to a small subpopulation of intracellular target proteins. Recent results indicate UCRP conjugation to target proteins proceeds through a ligation pathway distinct from that of ubiquitin. This novel mechanism for interferon action will be examined in six specific aims. (1) Physical characterization of UCRP-- the stability of recombinant UCRP and its precursor to various structural perturbants will be monitored by CD and fluorescence quench for comparison to ubiquitin. (2) Examine the binding of UCRP to intermediate filaments--transient expression of UCRP-chloramphenicol aminotransferase (CAT) in cultured A549 cells will be used to confirm earlier immunohistochemical data suggesting UCRP serves as a trans acting binding determinant for noncovalent association of target proteins with intermediate filaments. Deletion analysis of UCRP-CAT constructs will be utilized to identify the binding motif on UCRP responsible for filament association. Partial microsequencing will be used to characterize three novel low molecular weight (15-17 kDa) intermediate filament-associated proteins found to bind UCRP. (3) Examine the dynamics of intracellular UCRP pools--direct assays and complementation studies of (125)1-UCRP conjugation in cultured A549 cell extracts will examine the regulation of the ligation reaction during interferon-beta induction. Potential roles for UCRP in the interferon response will be tested by blocking UCRP synthesis through transient expression of antisense UCRP MRNA. (4) Purify and characterize the preUCRP processing activity. (5) Examine the enzymology of UCRP conjugation--in vitro (125)1-UCRP conjugation assays will be employed to identify the enzymes(s) present in A549 extracts catalyzing polypeptide ligation. These enzymes will be purified and characterized for comparison to the ubiquitin conjugation pathway. (6) Cloning of the UCRP conjugating enzymes--partial microsequencing of the UCRP conjugating enzymes will be used to construct probes for screening an interferon-induced A549 cDNA library. Cloning and expression of the UCRP conjugating enzymes for subsequent mechanistic studies will provide functional comparisons to the parallel ubiquitin ligation pathway.

描述（根据申请人的摘要改编）：特定于单元线 干扰素的效果通过协调的诱导介导 在细胞因子期间精确表达的基因子集 回复。 首席研究人员表明，p15，早期17 kDa 1型干扰素诱导的基因产物，与与 串联二 - 泛素序列。 随后的证据表明 这种泛素交叉反应性蛋白（UCRP）的生物反应是 通过共价连接介导的 细胞内靶蛋白。 最近的结果表明UCRP结合 靶向蛋白质通过不同的结扎途径进行 泛素。 这种干扰素作用的新型机制将是 在六个特定目标中进行了检查。 （1）UCRP的物理表征 - 重组UCRP及其前体的稳定性 结构扰动者将通过CD和荧光淬火监测 与泛素进行比较。 （2）检查UCRP与 中间丝 - UCRP-氯平苯基的透视表达 培养的A549细胞中的氨基转移酶（CAT）将用于确认 较早的免疫组织化学数据表明UCRP用作反式 靶蛋白非共价关联​​的作用结合决定因素 带有中间细丝。 UCRP-CAT构建体的删除分析 将利用用于确定ucrp的绑定基序 细丝协会。 局部微钉将用于 表征三个新型的低分子量（15-17 kDa）中间体 发现与UCRP结合的丝相关蛋白。 （3）检查 细胞内UCRP池的动力学 - 独立测定和补充 （125）1-UCRP在培养的A549细胞提取物中的结合将 检查干扰素β过程中连接反应的调节 就职。 UCRP在干扰素反应中的潜在作用将是 通过通过瞬态表达的瞬时表达来测试 反义UCRP mRNA。 （4）纯化并表征Preucrp处理 活动。 （5）在体外检查UCRP结合的酶学 （125）将使用1-UCRP共轭测定法来识别 A549提取物中存在的酶催化多肽连接。 这些酶将被纯化并进行特征，以进行比较 泛素结合途径。 （6）UCRP连接的克隆 UCRP共轭酶的酶 - 派对微钉将 用于构建探针以筛选干扰素诱导的A549 cDNA库。 UCRP共轭酶的克隆和表达 对于随后的机械研究，将提供功能比较 到平行的泛素连接途径。